The secretin receptor is prototypic of the important Class B family of G protein-coupled receptors. The long-term goal of this work is to better understand the structure, function, and regulation of this group of receptors, gaining insights that will facilitate the development of new therapeutic strategies and new drugs that can act at these targets. The projects are designed to test, extend, and refine the recently proposed molecular model of the natural agonist-occupied secretin receptor and to elucidate the molecular basis of receptor activation and receptor regulation by oligomerization within the plasma membrane. There are three broad aims for this proposal. The first aim is designed to explore the hypothesis that the amino-terminal domain of the secretin receptor provides a critical ligand-binding pocket that undergoes a conformational rearrangement upon binding the natural peptide agonist. This will be investigated by photoaffinity labeling specific sites within the receptor using series of agonist and antagonist probes, by developing and applying fluorescent indicators within agonist and antagonist probes, and by the application of fluorescence resonance energy transfer techniques. The second aim is designed to explore the molecular mechanism of transduction of the activation signal from the receptor amino terminus to the receptor body, examining the novel hypothesis predicting the presence of an endogenous agonist within the receptor sequence that is exposed upon agonist binding. This will be examined by photoaffinity labeling with charge-modified ligands, site-directed receptor mutagenesis, and biological activity studies using synthetic candidate molecules. The third aim is designed to examine the molecular basis and functional importance of secretin receptor oligomerization as a mechanism to regulate the secretin receptor in health and disease. This will be examined using bioluminescence resonance energy transfer with modified receptor constructs, studying impact on function and receptor association. In addition to wild type receptor, a misspliced variant of the secretin receptor recently described in various neoplasms that has dominant negative inhibitory activity will also be studied. Together, these efforts should provide the finest level of molecular detail available for understanding the structure and mechanisms of ligand binding, activation, and regulation of any receptor in this receptor family.